Cellulose derivatives

ABSTRACT

Stable microdispersed polyanhydroglucuronic acid and salts thereof contain in their polymeric chain from 8 to 30 percent by weight of carboxyl groups, at least 80 percent by weight of these groups being of the uronic type, at most 5 percent by weight of carbonyl groups including intra- and inter-molecular 2,6 and 3,6 hemiacetals, 2,4-hemialdals and C2 and C3 aldehydes. The molecular mass of the polymeric chain is from 1×10 3  to 3×10 5  Daltons. The microdispersed products are prepared by subjecting a polyanhydroglucuronic acid-containing material to partial or complete hydrolysis and neutralisation in an oxidative environment.

[0001] The present invention relates to a stable microdispersedpolyanhydroglucuronic acid (PAGA) and salts thereof, especially suitablefor medicinal, pharmaceutical and cosmetic products, as well as to amethod of preparing the same. The term polyanhydroglucuronic acid andsalts thereof as used herein includes copolymers thereof, especiallywith anhydroglucose.

INTRODUCTION

[0002] Besides proteins, polysaccharides represent the most widespreadbiopolymers found in the biosphere. As an example, up to 10¹² metrictonnes per year of cellulose, a 1, 4 β D-glucane, is synthesized innature Other α and β glucanes bound e.g. by 1,2; 1,3; 1,4 and 1,6; or1,2 and 1,4 glycosidic bonds in the main chain, mostly of microbialorigin, gam increasing importance with ongoing research in the field Itis the presence of glucuronic acid units in the polymeric chain of theoligosaccharides or polysaccharides that, together with their molar massand type of the principal glycosidic bond, constitutes the basis oftheir immunostimulative, antitumourous, anticoagulative, or elsehaemostyptic effects (cf. Burchard W. Ed., Polysaccharide, Eigenschaftenand Nutzung, Springer Verlag, Berlin 1985, p. 144).

[0003] Glucuronoglucanes can preferably be prepared by relativelyspecific selective oxidation of the primary alcoholic group at C6 carbonatom of the glucopyranosic unit of natural polysaccharides by nitrogenoxides, the C1 aldehydic group of the basic unit being protected by theglycosidic bond.

[0004] A variety of methods have been disclosed for preparingglucuronoglucanes and glucuronanes from natural glucanes, using theoxidative effects of NO_(X) either in the gaseous form (Kenyon et al.,Ind. Eng. Chem., 41, No 1, 2-8 (1949); DE 0941289; DE 0967144), innonpolar reaction environment of inert liquids such as hydrogenatedhydrocarbons (USSR SU 937462; U.S. Pat. No. 4,347,057; EP 0492990), orin polar environment of aqueous solutions of acids such as HNO₃, H₃PO₄or their mixtures with HSO₄, wherein the NO_(X) are mostly generateddirectly in the oxidation liquor via dosed introduction of reducingsubstances such as, notably, NaNO₂ (GB 709684; CS AO 185366; GB 1593513;Painter J. et al., Carbohydrate Research 140, 61 (1985); Alhaique F.,Chim. Oggi 11-15, 17 (1986)), or the reaction environment is created byintroducing liquid NO_(X) into aqueous HNO₃ (U.S. Pat. No. 4,100,341).

[0005] A disadvantage of these known processes relates to the fact thattheir oxidative effects on the glucane molecule are non-uniform and onlyrelatively specific in that besides creation of carboxyl groups of theuronic type of C6 carbon of the glucopyranosic unit, other types ofsuccessive reactions (such as formation of ONO₂ and NO groups on C6) andsecondary reactions (such as formation of COOH and other oxidised groupson end carbons C1 and C4, and notably on C2 and C3 carbons) do occur. Inaccord with numerous publications (Kaversneva E. P., Doklady AN SSSR(U.S.S.R.) 78 (3), 481 (1951); Nevell T. P., J. Text. Ind. 42, 91(1951); Sihtola M. et al., J. Polym. Sci, Part C, (2), 289 (1963);Pastéka M., Chemické Zvesti (Slovakia) (20), 855 (1966)), extensivetesting of polyanhydroglucuronic acids prepared by the action of NO_(X)has led us to the conclusion that, besides carboxyl groups on C6 carbon,several other aldehydes, ketones, and their condensation products areformed that have fuidamental influence on the stability of thepolyanhydroglucuronic acid product

[0006] It is known that the presence of carbonyl groups can be limitedby their back reduction to primary alcoholic groups by means of complexhydrides such as NaBH₄ (Charkin S. W. and Brown W. G., J. Am. Chem. Soc.71, 122 (1949); Mead F. S. M., J. Text, Inst. 46, T 400 (1955)), butthis process is quite expensive for industrial use due to the cost ofthe hydrides.

[0007] The quality of the product also depends on both the input rawmaterial and the technological method used. Natural glucanes occur inthe form of fibres, globules or grams with varying degree of orderliness(crystallinity). Their oxidation and partial degradation due to theeffect of NO_(X) does not proceed with the same speed in crystalline andamorphous regions, so that the resulting product represents a mixture ofmacromolecules oxidised and degraded to various extents which mayprovide products which are physiologically ineffective and/or havenegative effects.

[0008] It is evident from the above that the preparation of stable PAGAproduct having required physical and chemical characteristics, destinedfor pharmaceutical and cosmetic use, is in no way a simple matter.

[0009] In health care practice one often encounters cases of capillarybleeding occurring during injuries or related to surgical interventions.The healing of the wounds frequently depends on attaining rapidhomeostasis and creation of coagulum, to especially serve as aprotection of the wound against infection. Application of Dglucurono-1,4 β D-glucane, the so-called oxidised cellulose, as anon-toxic resorbable local haemostatics to arrest bleeding from surfaceinjuries or parenchymatous organs, osseous bleeding, and in generalwherever use of conventional styptic means may be difficult or slow infunctioning and less effective, has proved especially effective insimilar cases.

[0010] Experience has shown that the product should be stored attemperatures not exceeding 25° C., preferably below 10° C., protectedagainst direct light. When these conditions are not met, the influenceof light and/or elevated temperatures during storage may easily provokedegradation changes due to the instability of secondary reactive groupsand, on nitrogen-containing sites This in turn may be manifested byreduced tissue tolerability, and even virtually exclude application ofthe conventional product in some pharmaceutical or cosmetic preparations

[0011] In summary, methods of preparing PAGA known thus far are based onoxidative action of NO_(X) on suitable types of polysaccharides ofcellulosic or microbial origin (such as scleroglucanes), possibly withsubsequent reduction of the content of destabilizing groups viareduction by hydrides, the latter process being, however, relativelyexpensive and jeopardising the product with simultaneous reduction ofthe carboxyl group content via reduction of their carbonyls. No methodhas been found up to now for preparing stable polyanhydroglucuronic acidwith broader application scope enabling a better control of the finalproduct characteristics.

[0012] Among important disadvantages of the known methods quoted aboveare non-uniform degree of both oxidation and degradation of individualpolysaccharide particles or fibres, non-uniform content of boundnitrogen and other destabilizing sites in the macromolecule, as well asbroad distribution of their molecular masses, altogether factors whichcan result in non-uniformity in resorbtion in the organism on applyingthe product as a haemostatic or in binding other substances or drugssuch as anaesthetics, antibiotics or cytostatics.

[0013] In the latter case of active substance-PAGA complexes, thepresence of destabilizing groups in this otherwise importantbiologically degradable carrier brings about inherent instability andchanges in properties with time. The same applies to formulations forpharmaceutical or cosmetic use, for which our testing has revealeddiscoloration with temperature and time, viscosity changes, and evenphase separation, whenever unstabilized PAGA prepared by known methodswas utilised.

[0014] A further deficiency of the known methods lies in the fact thatPAGA prepared by NO_(X) oxidation displays closed surface and low valuesof specific surface area (measured in m².g⁻¹) for both fibrillar orparticulate material. Whenever final product in powder form is required,the isolated bulk product has to be mechanically disintegrated, in a dryor wet process, which brings about potential contamination by impuritiessuch as metals due to abrasion of production equipment and increasesfurther the production costs.

[0015] A last but not least disadvantage of the conventionally preparedPAGA products is that, in contrast to e.g. hyaluronic or algic acids,they do not allow conversion to a range of forms for differentapplications.

[0016] Some of the above deficiencies had been addressed by JBriestensky et al.)CS AAO 242920) who disclose an oxidisedcellulose-based sorbent consisting of highly porous non-agglomeratedparticles of PAGA, and a method of manufacturing the same, involvingtransformation of oxidised cellulose into a colloidally dispersed systemwith simultaneous partial hydrolysis followed by coagulation andstabilisation.

[0017] However, the above issues relating to the inherent structuralnon-uniformity of the raw oxidised products and its long-termdestabilising effects remain to be unsolved.

[0018] There is therefore a need for a method of preparing stablemicrodispersed polyanhydoglucuronic acid and salts thereof so that theproduct may be used in medicinal, pharmaceutical and cosmeticformulations.

STATEMENTS TO INVENTION

[0019] According to the invention there is provided a method forpreparing a product comprising polyanhydroglucuronic acid and/or saltsthereof wherein a polyanhydroglucuronic acid-containing materialobtained by oxidation with nitrogen oxides is subjected to partial orcomplete hydrolysis and neutralisation in an aqueous solution ofinorganic and/or organic salts and/or bases in the presence of suitableoxidising agents, the hydrolysate undergoing fractional coagulation toform a stable microdispersed/microdispersable product. The termpolyanhydroglucuronic acid and salts thereof includes copolymersthereof, especially with anhydroglucose.

[0020] This method provides stable polyanhydroglucuronic acid and saltsthereof in essentially a single process carried out in a single vessel.

[0021] Most preferably the inorganic and/or organic salts and/or basesused for hydrolysis are chlorides, sulphates, carbonates, formates, oracetates of alkali and/or alkaline earth metals, hydroxides of alkaliand/or alkaline earth metals, alkylamines, or alkanolamines, inconcentrations ranging from 1 to 10⁻³ to 5 mol/l.

[0022] In an especially preferred embodiment of the invention theoxidative environment during hydrolysis is established by the presenceof oxidising agents selected from one or more of hydrogen, sodium ormagnesium peroxide, peroxoacids and their salts, hypochlorites andchlorites.

[0023] Preferably the hydrolysate is let to undergo fraction coagulationby a suitable water-miscible organic solvent, the coagulated product iswashed, or dehydrated, using a suitable water-miscible organic solvent,and/or converted, in an appropriate manner, for intended subsequent use.

[0024] Preferably the procedure is carried out at a pH of from 1 to 12,and preferably, at a temperature of from 0 to 100° C.

[0025] In a preferred embodiment of the invention thepolyanhydroglucuronic acid-containing material is obtained by oxidationof a suitable polysaccharide, such as native or regenerated cellulose orstarch.

[0026] The invention also provides stablemicrodispersed/microdispersable polyanhydroglucuronic acid and saltsthereof wherever made using the method of the invention. In particular,the invention also provides novel stable microdispersedpolyanhydroglucuronic acid and salts thereof containing in theirpolymeric chain from 8 to 30 percent by weight of carboxyl groups, atleast 80 percent by weight of these groups being of the uronic type, atmost 5 percent by weight of carbonyl groups, and at most 0.5 percent byweight of bound nitrogen.

[0027] Preferably the product contains at most 0.2 percent by weight ofbound nitrogen in the polymeric chain.

[0028] In a preferred embodiment of the invention the molecular mass ofthe polymeric chain is from 1×10³ to 3×10⁵ Daltons, most preferably from5×10³ to 1.5×10⁵ Daltons.

[0029] The content of the carboxyl groups is in the range of from 12 to26 percent by weight and at least 95 percent of these groups are of theuronic type.

[0030] In a particularly preferred embodiment of the invention theproduct contains at most 1 percent by weight of carbonyl groups.Typically the carbonyl groups are intra- and intermolecular 2,6 and 3,6hemiacetals, 2,4-hemialdals and C2-C3 aldehydes.

[0031] The polyanhydroglucuronic acid and salts thereof may be made upof particles sized from 0.1 to 100 μm and/or fibres of from 5 to 30 μmdiameter and up to 30 mm length.

[0032] Because neutralisation and refining is carried out in a singleoperation the process is cost effective.

[0033] As the product is in a microdispersed form there is enhancedsorption and greater accessibility for blood. Therefore the biologicalavailability is increased and a rapid onset of haemostasis. We have alsoobserved that the product assists wound healing as a large surface areais presented which is quickly penetrated by body fluids and goes intosolution in these fluids. We believe that the product then chemicallydegrades to achieve more rapid absorption and enhancement of the woundhealing process.

[0034] The overall homogeneity of the distribution of oxidised groupswithin the product is increased. Thus, the product has improvedreactivity and accessibility to reactive sites for the purpose ofbinding other substances such as pharmacologically active substances tothe product. The average degree of polymerisation is decreased, thedistribution of the polymerisation is narrowed and the amount ofcellulosic fractions are reduced. This also assists in biodegradation.

[0035] The products of the invention are notably free or hydratedaldehydic groups on C2 and C3 carbons of the basic unit, their intra-and intermolecular hemiacetals, intramolecular C2, C6 hemiacetals,intermolecular C2 and C3 hemialdals, and monoketonic groups on C2 and C3carbons. Presence of even small amounts of these groups may destabilizemain glycosidic bonds and result in formation of irritating products,especially in applications in aqueous systems.

[0036] In a final stage of the degradation process after oxidation andisolation of the product during its storage, macromolecular products maybe formed which are physiologically ineffective or even have irritatingor other negative effects on the organism. In addition we have foundthat equally undesirable from both the physiological and stabilitystandpoint is the content of bound nitrogen, albeit in smallconcentrations, mostly occurring in the form of nitrosoether or nitritegroups. These groups may undergo scission leading to formation ofnitrogen containing acids which in turn may provoke destruction of thePAGA product during storage.

[0037] The invention also provides a pharmaceutical or cosmeticcomposition incorporating stable microdispersed polyanhydroglucuronicacid and salts thereof of the invention.

[0038] The invention will be more clearly understood from the followingdescription thereof given by way of example only.

DETAILED DESCRIPTION

[0039] It has been our aim to prepare stable polyanhydroglucuronic acidwith controlled physicochemical properties adapted to the intended use,thus reducing or fully suppressing deficiencies of conventional productsmanufactured as well as broadening the potential scope of applicationsthereof. This aim is achieved by preparing stabilized microdispersedPAGA with reduced degree of crystallinity, its copolymers withanhydroglucose, and salts thereof, with a high degree of purity

[0040] An important feature of the invention resides in that thismicrodispersed PAGA, its copolymers with anhydroglucose, and saltsthereof, prepared according to the invention, comprising a reducedproportion of the crystalline phase, consists of particles of 0.01 to1000 μm in size or fibres with 5 to 30 μm diameter and up to 30 mmlength, with an open surface, containing in their polymeric chain from 8to 30 percent by weight of carboxyl groups, at least 80 percent of thesegroups being of the uronic type, and a reduced proportion ofdestabilizing carbonyl groups, in particular aldehydic ones on C2 and C3carbons of the basic glucopyranosic unit and condensation productsthereof, notably intra-and intermolecular 2,6- and 3,6-hemiacetals,2,3-hemialdals and C2-C3 aldehydes, as well as of bound nitrogen.

[0041] Aiming at suppression of the above mentioned deficiencies,especially of low stability, of the PAGA products manufactured thus far,as well as of deficiencies of known methods of preparing the same, isalso the method of preparing according to the invention, which yieldsstable microdispersed PAGA with easily controllable physicochemicalcharacteristics. An important feature of the process consists in thatthe raw PAGA product obtained by oxidation of a suitable type of naturalglucane and cleared, e.g. by washing, of foreign admixtures istransformed, via action of aqueous solutions of salts such as sodiumacetate or carbonate or calcium acetate, chloride or carbonate and/ororganic or inorganic bases such as alkyl- or alkanolamines or alkalimetals or alkaline earth hydroxides, within an oxidative environmentconstituted by e.g. organic or inorganic peroxides and/or peroxoacidsand salts thereof or hypochlorites or chlorites, into an aqueouscolloidal dispersion system, simultaneously provoking hydrolysis oforiginal macromolecular chains of PAGA, oxidation of the destabilizingcarbonyl groups in the original PAGA to stable carboxyl groups, andhydrolytic removal of bound nitrogen, whereupon the reaction system iscoagulated and stabilised by means of a water-miscible coagulatingagent, separated microdispersed PAGA or a salt thereof Is washed,isolated, and dehydrated using a water-miscible solvent such as C1 to C4monohydric aliphatic alcohol, or else first modified by some of knownphysical or chemical methods and then washed, isolated, and dehydratedin much the same way.

EXAMPLES Example 1

[0042] In this example, the raw material for preparing a salt ofmicrodispersed polyanhydroglucuronic acid were cotton linters containing99.1% b/w (by weight) of α-cellulose and oxidised in 60% nitric acidwith an admixture of 3.6% nitrous acid at a temperature of 28° C. inanalogy with the procedure of GBP 709684. The resulting productcontained:

[0043] carboxyl groups . . . 13.7% b/w

[0044] carbonyl groups . . . 4.2% b/w

[0045] bound nitrogen . . . 0.48% b/w

[0046] In a 3000 ml laboratory mixer, 1000 ml of water and 0.158 g ofcalcium acetate were heated up to 60° C. and stirred at 600 rpm. Afterdissolution of calcium acetate, 2 g of the above defined oxidised cottonlinters containing about 8% of volatile matter were added, temperatureincreased to 98° C., and the mixture stirred at 2800 rpm for 15 minuteswhile maintaining the temperature. The temperature was then decreasedback to 60° C., pH adjusted to 8.5 by adding sodium hydroxide solution,25 g of 30% hydrogen peroxide were added, and the hydrolysis continuedat the reduced temperature for another 15 minutes Subsequently thereaction system was cooled down to 40° C., stirring reduced to 300 rpm,and 1500 ml of 92% ethanol were added stepwise during about 10 minutesThe resulting colloid dispersion solution was then filtered, the residuewas dispergated into 50% water-ethanol mixture and allowed to stand forone hour. After another filtration the residue was redispergated into100 ml of isopropanol and allowed to stand for 6 hours. The sameprocedure was repeated once more, and then the product was filtered anddried in a vacuum drier at a temperature of 40° C.

[0047] An analysis of the product obtained yielded:

[0048] loss on drying . . . 1.25% b/w

[0049] carboxyl group content . . . 16.8% b/w

[0050] carbonyl groups . . . 0.5% b/w

[0051] bound nitrogen content . . . 0.13% b/w

[0052] calcium content . . . 2.1% b/w

[0053] sodium content . . . 5.2% b/w

[0054] particle size . . . 2 to 5 μm

[0055] specific surface area . . . 98 m²/g

[0056] Molecular weight . . . 6×10⁴ Daltons

[0057] The product can be used directly as a hemostatic powder or as acomponent of an aerosol powder spray.

Example 2

[0058] The raw material used was prepared via oxidation of a microbial(1→6)-βD-gluco-(1→3)-βD-glucane by gaseous nitrogen oxides, produced ina reaction of dried solid sodium nitrite with 75% b/w nitrous acid, andcontained:

[0059] carboxyl groups . . . 17.3% b/w

[0060] carbonyl groups . . . 1.3% b/w

[0061] bound nitrogen . . . 0.44% b/w

[0062] In a 1500 ml laboratory mixer, 500 ml of water and 80 g of theair-dried oxidised raw material were mixed together and stirred at 250rpm. 12 g of sodium peroxide were added gradually while strring. Thestirrer revolutions were then set at 1800 rpm and the system wasmaintained at a temperature of 52° C. for 30 minutes. The temperaturewas then decreased to 35° C., stirrer set at 120 rpm, pH adjusted to 6.0by adding sodium hydroxide solution, 480 ml of 92% ethanol were added,and the system stirred at 800 rpm for 5 minutes. Subsequently thereaction system containing a colloid dispersion of sodium salt ofD-glucurono-(1→6)-βD-gluco-(1→3)-βD-glucane was filtered and the residuewas dialysed for 4 days against pure water. The dialysed product wasredispergated into 500 ml of 80% ethanol and filtered again. The residuewas redispergated into 400 ml of isopropanol and allowed to stand for 24hours. Finally, the product was filtered and dried in a vacuum drier ata temperature of 40° C.

[0063] 62 g of the product were obtained with the followingcharacteristics:

[0064] loss on drying . . . 2.3% b/w

[0065] carboxyl group content . . . 18.9% b/w

[0066] carbonyl groups . . . 1.1% b/w

[0067] bound nitrogen content . . . <0.1% b/w

[0068] calcium content . . . 2.1% b/w

[0069] sodium content . . . 6.99% b/w

[0070] particle size . . . 10 to 20 μm

[0071] specific surface area . . . 28 m²/g

[0072] Molecular weight . . . Daltons

[0073] The product can be used directly as a hemostatic powder, possiblyas an active component of an aerosol formulation, and/or possibly as anactive component in cosmetic formulations and the like.

Example 3

[0074] Maize starch was processed by oxidation with gaseous nitrogenoxides at a temperature of 15 to 28° C. The intermediate product servingas the raw material in this example contained:

[0075] carboxyl groups . . . 23.8% b/w

[0076] carbonyl groups . . . 8.2% b/w

[0077] bound nitrogen . . . 0.62% b/w

[0078] volatile matter . . . 11.1% b/w

[0079] 15 g of the above defined oxidised raw material was dispergatedby stirring at 600 rpm in 200 ml of water using a 750 ml laboratorymixer. 11 g of sodium hypochlorite were added gradually, pH adjusted toa value of 2.3 by addition of hydrochloric acid, and the hydrolysis waslet to occur for 60 minutes at a temperature of 35° C. The pH value wasthen set at 7.5 by adding sodium hydroxide solution. The stirrer wasthen set at 2200 rpm and the reaction continued for another 15 minutes.Afterwards, 250 ml of 95% ethanol were added while stirring at 600 rpmfor 30 minutes at a temperature of 25° C. The product was thencentrifuged, dispergated into 70% ethanol, again centrifuged,redispergated into isopropanol, and allowed to stand for 24 hours. Thelatter operation was repeated once again, the product filtered and driedin a vacuum drier at 60° C.

[0080] An analysis of the product obtained yielded:

[0081] loss on drying . . . 34% b/w

[0082] carboxyl group content . . . 26.9% b/w

[0083] carbonyl group content . . . 1.8% b/w

[0084] bound nitrogen content . . . 0.18% b/w

[0085] sodium content . . . 11.5% b/w

[0086] particle size . . . 5-15 μm

[0087] specific surface area . . . 75 m²/g

[0088] Molecular weight . . . 92,000 Daltons

[0089] The product can be used for analogous purposes as that of Example2.

Example 4

[0090] Medicinal cotton was oxidised in a liquid system involving 3.9%b/w of nitrous acid in 65% nitric acid at temperatures between 3 to 28°C. The intermediate product serving as the raw material in this examplecontained:

[0091] carboxyl groups . . . 24.5% b/w

[0092] carbonyl groups . . . 6.9% b/w

[0093] bound nitrogen . . . 0.58% b/w

[0094] volatile matter . . . 9.9% b/w

[0095] A 1500 ml laboratory mixer was filled with 700 ml of water, and0.4 g of zinc chloride, 11 g of magnesium nitrate hexahydrate, 35.5 g ofcalcium chloride, and 32 g of disodiumcarbonate were added graduallyunder permanent stirring. A white emulsion of the salts was heated to40° C. while stirring at 150 rpm. Subsequently, 115 g of the oxidisedmedicinal cotton as described above were added and the stirringcontinued for another 10 minutes at 40° C. The hydrolysis was thencontinued with addition of 300 g of 10% solution of peracetic acid foranother 10 minutes. The system was agitated at 900 rpm for 30 seconds,cooled down to 20° C., and the fibrillar suspension filtered to removethe liquid. The residue was transferred to a 8000 ml sulfonation flask,suspended in 600 ml of 17% isopropanol/water mixture, and pH value ofthe system was adjusted to 6.0 by adding 10% solution ofcalciumdihydroxide in a 8% water solution of saccharose. The suspensionwas then filtered on a vibrating filter partition, resuspended in 17%isopropanol/water mixture and filtered again. Subsequently, the residuewas repeatedly washed with isopropanol and filtered. The residual fibrelayer in the form of a nonwoven mat was then dried in a vacuum drier at50° C.

[0096] The procedure yielded 85 g of the product with the followingcharacteristics:

[0097] loss on drying . . . 2.6% b/w

[0098] carboxyl group content . . . 28.4% b/w

[0099] carbonyl group content . . . 0.9% b/w

[0100] calcium content . . . 9.5% b/w

[0101] zinc content . . . 0.10% b/w

[0102] magnesium content . . . 0.41% b/w

[0103] sodium content . . . 1.8% b/w

[0104] bound nitrogen content . . . 0.11% b/w

[0105] fibre diameter . . . 10 to 18 μm

[0106] fibre length . . . 5 to 28 mm

[0107] specific surface area . . . 29 m²/g

[0108] Molecular weight . . . 1.5×10⁵ Daltons

[0109] The product may be used, after appropriate processing, formanufacture of modified wound dressings and similar products withhemostatic effects.

Example 5

[0110] Medicinal cotton gauze, alkali-bleached, was oxidised in a liquidsystem involving 2.8% b/w of nitrous acid in 67% nitric acid attemperatures between 5 to 15° C. The intermediate product serving as theraw material in this example contained:

[0111] carboxyl groups . . . 8.4% b/w

[0112] bound nitrogen . . . 0.72% b/w

[0113] volatile matter . . . 7.5% b/w

[0114] A 5000 ml laboratory mixer was filled with 1500 ml of water, and300 g of sodium hydroxide were added gradually under permanent coolingand stirring at 120 rpm. After dissolution the temperature was held at5° C., and 325 g of the oxidised raw material as described above wereadded under permanent stirring. On completion the stirrer was set at 350rpm and the temperature maintained at 5 to 8° C. for 15 minutes. Thestirrer was slowed down again to 120 rpm, and still while cooling, thevalue of pH was set at 8.0 by adding hydrochloric acid. The temperaturewas then increased to 20° C. and 200 g of 30% hydrogen peroxide wereadded. Subsequently, the system was stirred at 600 rpm for 20 minutes,1000 ml of concentrated ethanol were added and the stirring continuedfor another 10 minutes. The product was filtered, redispergated into1500 ml of 30% ethanol/water mixture, and agitated for 2 hours. The sameprocedure was then repeated another two times. Finally, the product wasredispergated in 1500 ml of methanol, allowed to stand for 6 hours,filtered, and redispergated once more in 1000 ml of isopropanol.

[0115] The procedure yielded the product in the form of a microfibrillarsuspension with the following characteristics:

[0116] carboxyl group content . . . 28.4% b/w

[0117] carbonyl group content . . . <0.10% b/w

[0118] sodium content . . . 5.9% b/w

[0119] bound nitrogen content . . . <0.10% b/w

[0120] fibre diameter . . . 10 to 15 μm

[0121] fibre length . . . 1 to 10 mm

[0122] Molecular weight . . . 1.5×10⁵ Daltons

[0123] The product can be used, after appropriate processing, formanufacture of modified wound dressings and similar products withhemostatic effects.

[0124] An important aspect of the invention consists in the ability ofthe microdispersed polyanhydroglucuronic acid and salts thereof to formstable dispersions in physiologically indifferent liquids displaying lowto zero rate of sedimentation, low viscosity of these colloid-dispersionnon-aqueous systems, and no tendency to agglomerate, at concentrationsof 0.5 to 15% b/w.

[0125] Of important advantage is the fact that the physicochemicalproperties of the microdispersed polyanhydroglucuronic acid can becontrolled to fit the dispergating liquid or mixture of liquids, thusallowing stable systems suitable as spray fillings to be prepared.

[0126] Extensive tests have shown that the microdispersedpolyanhydroglucuronic acid and salts thereof prepared by controlledhydrolysis and fractionation, mostly in the form of particles smallerthan the size of an erythrocyte, is capable of stimulating the activityof histiocytes and macrophages, which represents another essentialadvantage of the application of such substances. At the same time, theyeffectively arrest capillary bleeding of the wound area while gettingincorporated into the fibrin net formed. Due to small size of the orderof microns, the particles of the microdispersed polyanhydroglucuronicacid and salts thereof undergo, dependent on the chemical compositionand physiochemical properties, relatively rapid enzymatic hydrolysis inthe wound environment yielding glucose and glucuronic acid, substancesinherent to living organisms, as final products; in fact, histologicalobservations indicate that they are presumably incorporated into bodymucopolysaccharides. Close to neutral pH value of extracts of salts ofmicrodispersed polyanhydroglucuronic acid also substantially contributesto their biocompatibility; no adverse secondary effects due to acidicnature have been reported in their applications.

[0127] The presence of reactive carboxyl groups in the microdispersedpolyanhydroglucuronic acid and salts thereof is the basis for theirability to chemically bind substances with antibacterial effects such ase.g. derivatives of biguanid, quaternary ammonium salts, oraminosaccharide based antibiotics. Bactericidal activity is alsoobserved for salts or complex salts of certain cations, such as Zn²⁺,Cu²⁺, and to a limited extent Ag⁺, with microdispersedpolyanhydroglucuronic acid.

[0128] Similarly, we have observed that preparations based on themicrodispersed polyanhydroglucuronic acid and salts thereof displaycertain insecticidal activity. This activity can be enhanced usinghydrophobic reactivity of polyanhydroglucuronic acid molecules whichallows to anchor, on the powder substance, non-toxic syntheticderivatives of natural pyrethrins such as pyrethroids, notablyPermethrin (cis/trans isomer ratio 1:3). Another advantage ofcompositions according to the invention is thus represented by thepossibility to combine, in a single product, hemostatic, bacteriostatic,and insecticidal function. This is important in veterinary medicine forthe treatment of both traumatic and artificial lesions in e.g. sheep andcattle, in that it provides a temporary protection against microbialinfection and insect attack during healing.

[0129] An example of successful combination of an antibiotic andhemostatic may be represented by the application of neomycine ut sulfasand bacitracinum zincicum bound to a sodium/calcium salt ofmicrodispersed polyanhydroglucuronic acid.

[0130] Another problem that had to be solved within the inventionconcerns the choice of dispergating liquids and propellants to be usedin aerosol packaging formulations of the microdispersedpolyanhydroglucuronic acid and salts thereof.

[0131] Extensive tests have surprisingly revealed that the use oforganosols containing several different substituents or highly polarsubstituents caused the system to easily form coacervates or even tocoagulate.

[0132] We have found that eg alcoholic dispersions display a relativelylow stability with a rapid coagulation and/or sedimentation ofparticles. The stability is increased with increasing size of thealiphatic chain of the molecule, but the application of higher alcoholsis limited from the physiological point of view. We have also found thatthe hemostatic efficacy of the microdispersed polyanhydroglucuronic acidbased products in the initial phase immediately after the sprayadministration is reduced by the presence of water orpolyhydroxycompounds such as glycerol and its derivatives, glycols andpolyglycols. Univalent alcohols such as ethanol can induce a stingingpain on application to the wound. Substances of the latter types aretherefore preferably avoided in the formulation.

[0133] Coagulation and/or sedimentation was surprisingly equallyobserved in systems where a substance with low polarity has been used,but the molecule contained several different substituents giving rise toan electrostatic non-equilibrium, the Examples of there beingdichlorotetrafluoroethane or trichlorofluoromethane. In contrast, lowpolar substances such as alkanes, C1 to C8 cycloalkanes, or theirfluorinated and perfluorinated derivatives, yielded stable dispersionsystems with a low sedimentation rate. Examples are methane, ethane,propane, butane, isobutane, pentane, 2-methylbutane, 2-methylpropane,2,2-dimethylpropane and the like. Substances with 3 to 5 carbon atomssuch as pentane, neopentane, or a pure petrol fraction free frommercaptanes and aromatics may preferably be used to reduce loss atadministration, to improve fixation of the substance upon the treatedarea.

[0134] We have further found that the organic liquid molecule may alsocontain a heteroatom, preferably oxygen, in the main chain withoutdeteriorating the system stability. Such substances would involve etherssuch as dimethylether, diethylether, but also perfluorinated ethers ofthe methoxy- or ethoxy-nonafluorobutane type.

[0135] Extensive tests have shown that the product, though involving animportant number of hydrophilic polar groups, can best be dispergated inlow polar or non-polar liquids with a low surface tension and lowrelative permitivity In contrast, we have found that liquids with higherpolarity and higher surface tension tend to support agglomeration of theproduct particles and thus to jeopardise the correct function of theaerosol packaging. Besides the effect of microparticles with a largespecific surface area, the good dispergability of the microdispersedpolyanhydroglucuronic acid and salts thereof may be attributed to theirability to enter, in spite of the presence of hydrophilic groups,hydrophobic interactions with the dispergating liquids. The resultsindicate that stable dispersion systems can preferably be obtained usingthose of the above substances which display a value of the relativepermitivity (dielectric constant at 25° C. and 10 kHz) less than 10,preferably less than 5, and that of the surface tension less than 30mN/m, preferably less than 15 mN/m. Thus the substances recommended foruse involve, preferably, C3 to C5 alkanes, isoalkanes, or cycloalkanes,1,1,1,2-tetrafluoroethane, dimethylether, methoxy- andethoxy-nonafluorobutane and mixtures thereof.

[0136] Besides the ability to form low sedimenting dispersion systems,the overall criteria limiting the choice of suitabledispergator/propellant systems further include: physiologicalindifference (low toxicity, zero or minimum skin and cardiacsensitisation at exposures up to 100000 ppm, no mutagenicity andcarcinogenicity, minimum solubility in water and body fluids),indifference in contact with the active substance, high volatility andlow heat of evaporation, ability to fix the active substance in thefirst phase immediately after application on the wound surface,environmental acceptability, and cost.

[0137] It is difficult to draw a sharp demarcation line between thedispergating medium suitable for the microdispersedpolyanhydroglucuronic acid and salts thereof and the propellant since insome cases both functions can be provided for by one and the samesubstance such as e.g. n-butane or isobutane. In general, the relevantsubstances may especially involve:

[0138] a) Aliphatic and alicyclic hydrocarbons with 1 to 6 carbon atoms,or aliphatic ethers, notably dimethylether, diethylether, anddiisopropylether. While aliphatic hydrocarbons with 1 to 3 carbon atomscould well serve as dispergators for the microdispersedpolyanhydroglucuronic acid and salts thereof when under pressure, theyevaporate immediately at the output of the spray outlet and thusincrease the powder dissipation on spraying and insufficiently fix thepowder on the wound surface. It is therefore preferable to use higherhydrocarbons such as n-butane, isobutane, n-pentane, or isopentane forthe given purpose. This group may also include petrolether,pentane/isopentane fraction from petroleum distillation, or a mixture ofliquid hydrocarbons currently distributed under the name of medicinalpetrol, under the obvious condition of being pure enough from aromatichydrocarbons and mercaptanes. From the ether group, dimethylether canpreferably be used with respect to its suitable physicochemicalcharacteristics.

[0139] b) Nonflammable compounds known as fluorohydrocarbons (HFC),perfluorocarbons (PFC), and recently introduced hydrofluoroethers (HFE).Compared to chlorofluorocarbons (CFC), the HFC's and PFC's display muchreduced life time in the atmosphere and zero to very low ozone-depletingpotential (ODP) and global warming potential (GWP). Some may have aslightly increased toxicity and bioreactivity; however, their contactwith the wound is very short due to the rapid evaporation rate. The mostsuitable choice with respect to the properties may be represented by1,1,1,2-tetrafluoroethane (HFC 134a), or hydrofluoro-ethers such asmethoxy-nonafluoroethane (HFE 7100) or1,1,1,2,3,3-hexafluoro-3-methoxypropane, ah of these substances beingacceptable from both the physiological and environmental point of view.Representatives of both above groups are liquids or substancesliquefiable at low pressures (0.2-1.4 Mpa) at normal conditions. Furtheralternatives include:

[0140] c) Gaseous substances, which cannot be liquefied at normalconditions, but capable of being absorbed, at least partially, in thepowder active substance or in the liquid dispersion system. Theseinclude notably carbon dioxide, and nitrous oxide.

[0141] d) Gaseous substances not liquefiable at normal conditions anddisplaying a very limited absorption ability in the liquid dispersionsystem, such as rare gases, air, and nitrogen.

[0142] All of these substances can further be suitably combined witheach other to provide for an optimized function of the spray. Based onextensive testing, the preferred combinations include systems such asn-butane or n-pentane/CO₂, medicinal petrol/HFC 134a,isopentane/dimethylether, medicinal petrol/HFE 7100/HFC134a, HFE7100/CO₂, n-pentane/HFE 7100/N₂.

[0143] In summary, the important fact underlying the present inventionis that the specifically prepared microdispersed polyanhydroglucuronicacid and salts thereof make it possible to create stable dispersionconcentrates in liquids that do not compromise the environment,displaying zero or low values of both the ODP and GWP potentials.

[0144] An important advantage of the aerosol packaged hemostaticaccording to the invention consists in the fact the contents of thepackaging can repeatedly be used without the loss of their sterility.The dosing of the active substance can accurately be directed to thewound surface where the powder gets well anchored due to the relativelyhigh speed of incidence of an indifferent dispersion in a liquid that isimmiscible with the body fluids and evaporates within a few seconds.

[0145] Although certain adverse secondary effects are reported for theabove listed dispergating and propellant substances, such as weaknarcotic effects or skin degreasing on contact for C5 hydrocarbons, nosuch effects have been observed during extensive application tests ofthe sprays according to the invention because of small applied amountsand short contact time.

[0146] An additional specific advantage can be attained when usingsubstances listed under a) above or combinations of substances listedunder a) and c) above for preparing the stable dispersions of themicrodispersed polyanhydroglucuronic acid and salts thereof. Suchformulations of the spray allow a simple terminal sterilisation of thefinished aerosol packagings to be performed by gamma radiation.

Example 6

[0147] A hemostatic composition in pressurised aerosol packaging hasbeen prepared using stable microdispersed polyanhydroglucuronic acid inthe form of calcium/sodium salt according to Example 1 above. Theequipment used included a stainless steel 1000 litre mixer with apropeller stirrer, a stainless steel 30 litre/min metering pump withinner circulation, and an aerosol filling machine (Pamasol type) withone filling head for the dispersion concentrate and two filling headsfor the propellant.

[0148] The bulk substance used in this example was a calcium/sodium saltof microdispersed polyanhydroglucuronic acid having the followingcharacteristics:

[0149] Particle size 20-60 μm . . . 2% b/w

[0150] 10-20 μm . . . 32% b/w

[0151] ≦10 μm . . . 66% b/w

[0152] specific surface area . . . 105 m²/g

[0153] Carboxyl group content (total) . . . 20.2% b/w

[0154] carboxyl group content (uronic) . . . 18.2% b/w

[0155] free formaldehyde . . . 0% b/w

[0156] foreign particles . . . 0% b/w

[0157] calcium content . . . 3.9% b/w

[0158] sodium content . . . 5.6% b/w

[0159] bound nitrogen content . . . 0.02% b/w

[0160] Chlorohexidine hydrochloride (Ferrosan) in concentration of 0.1%b/w was added as a bacteriostatic adjuvant. The dispergation/propellantsystem involved a liquid hydrocarbon mixture (known as medicinal petrol)with density of 652 kg/m³, boiling point 55° C., and residue afterevaporation <2 ppm, and 1,1,1,2-tetrafluoroethane (HFC 134a).

[0161] 40 kg of the active substance was placed into the mixer, 150litres of the liquid hydrocarbon mixture added, and the system stirredat 600 rpm for 5 minutes. After addition of 1 kg of chlorohexidinehydrochloride and of another 250 litres of the liquid hydrocarbonmixture, the system was further stirred until a uniform dispersion wasobtained. The metering pump was used to dose the dispersion via thefilling head of the filling machine into aerosol cans of 80 ml nominalvolume in doses of 31 g per can. After inserting a suitable valve,another filling head was used to add 18 g per can of the1,1,1,2-tetrafluoroethane propellant. The finished spray can be used fortreatment of bleeding wounds by both the professional or a layman.

Example 7

[0162] The same equipment was used as in Example 6. The active substanceconsisted of two components, MDOC1 with the same characteristics as inExample 6, and MDOC2 involving a zinc/calcium/sodium salt ofmicrodispersed polyanhydroglucuronic acid having the followingproperties:

[0163] carboxyl group content . . . 19.5% b/w

[0164] free formaldehyde . . . 0% b/w

[0165] zinc content . . . 9.5% b/w

[0166] calcium content . . . 3.9% b/w

[0167] sodium content . . . 5.6% b/w

[0168] bound nitrogen content . . . 0% b/w

[0169] Neomycinum ut sulfas and Bacitracinum zincicum were used asantibacterial adjuvants, n-pentane having density of 625 kg/m³, andboiling point 36° C., as the dispergator, and carbon dioxide (ediblegrade quality) as the propellant.

[0170] 38.8 kg of MDOC1 and 1.2 kg of MDOC2 was placed into the mixertogether with 0.132 kg of Neomycinum ut sulfas and 0.143 kg (10⁷ IU) ofBacitracinum zincicum, 200 litres of n-pentane added, and the systemthoroughly stirred. Another 200 litres of n-pentane were then added andstirred for another 10 minutes. Aerosol cans of 80 ml nominal volumewere then filled in doses of 31 g per can, and, after inserting thevalves, another filling head was used to pressurise the can by additionof 2 g of compressed carbon dioxide.

[0171] The finished spray can be used for professional treatment ofbleeding wounds and lesions.

Example 8

[0172] A thoroughly homogenised uniform powder mixture of microdispersedpolyanhydroglucuronic acid in the form of magnesium/calcium/sodium andzinc/calcium/sodium salts in the mass ratio of 32:1 is filled intoaerosol cans of 210 ml nominal volume in doses of 8 g per can on apowder dosing machine (Bosch). Upon closing the can with an appropriatevalve, the can is pressurised on the aerosol filing machine by adding 20g of n-butane and 30 g of dimethylether. Finished and gamma sterilisedsprays then can be used for treatment of smaller burns or scalds. It canalso be applied in e.g. urological or gynecological surgery.

Example 9

[0173] Into the mixer as described in Example 6, 25 kg of calcium/sodiumsalt of microdispersed polyanhydroglucuronic acid (Example 6), 5.0 kg ofcalcium stearate, 0.4 kg of chlorohexidine hydrochloride, and 7.7 kg ofPermethrin with cis/trans ratio of 25:75 (ICI Plant Protection) aresuccessively placed. After addition of 400 litres of n-pentane and 99 kgof methoxy-nonafluorobutane (HFE 7100), the contents of the mixer arestirred for 15 minutes. The uniform dispersion is then filled intoaerosol cans of 210 ml nominal volume in doses of 110 g per can on thefilling machine and closed with an appropriate valve. Another fillinghead is then used to add 50 g per can of dimethylether.

[0174] The finished spray is designed for use in veterinary practice fortreatment of wounds and lesions in e.g. sheep and cattle, simultaneouslyproviding a temporary protection against microbial and/or insect attack.

Example 10

[0175] A hydrophilised adduct (MDOC-ACV) of9-[(2-hydroxy-methoxy)-methyl]-guanin (acyclovir) and a calcium/sodiumsalt of microdispersed polyanhydroglucuronic acid (Example 6) wasprepared according to Example 1 above by a hydrolytic treatment withcontrolled pH and fractionation, the content of acyclovir in theMDOC-ACV adduct being 50.5% b/w

[0176] A homogenised mixture of the adduct, the magnesium/calcium/sodiumsalt of microdispersed polyanhydroglucuronic acid according to Example6, and the zinc/calclum/sodium salt thereof according to Example 7 inthe mass ratio of 8.7:0.3:1.0 is filled into aerosol cans of 120 mlnominal volume in doses of 4 g per can on a powder dosing machine(Bosch). Upon closing the can with an appropriate valve, the aerosolfilling machine is used to pressurise the can by adding 25 g of2,2-dimethylpropane (neopentane) with a density of 625 kg/m³, andboiling point 9.6° C., on filling head 1, and 23 g of dimethylether witha density of 668 kg/m³.

[0177] The finished spray is packed and sterilised by gamma radiationwith a dose of 25 kGy and used as a dusting powder in the treatment ofe.g. herpes zoster, enabling an easy application and good efficacy evenin hairy areas of the body.

[0178] The invention is not limited to the embodiments hereinbeforedescribed which may be varied in detail.

1. A method for preparing a product comprising polyanhydroglucuronicacid and/or salts thereof wherein a polyanhydroglucuronicacid-containing material obtained by oxidation with nitrogen oxides issubjected to partial or complete hydrolysis and neutralisation in anaqueous solution of inorganic and/or organic salts and/or bases in thepresence of suitable oxidising agents, the hydrolysate undergoingfractional coagulation to form a stable microdispersed product.
 2. Amethod as claimed in claim 1 wherein the inorganic and/or organic saltsand/or bases used for hydrolysis are chlorides, sulphates, carbonates,formates, or acetates of alkali and/or alkaline earth metals, hydroxidesof alkali and/or alkaline earth metals, alkylamines, or alkanolamines,in concentrations ranging from 1 to 10⁻³ to 5 mol/l.
 3. A method asclaimed in claim 1 wherein the oxidative environment during hydrolysisis established by the presence of oxidising agents selected from one ormore of hydrogen, sodium or magnesium peroxide, peroxoacids and theirsalts, hypochlorites and chlorites.
 4. A method as claimed in claim 1wherein the hydrolysate undergoes fractional coagulation by a suitablewater-miscible organic solvent.
 5. A method as claimed in claim 4wherein the coagulated product is washed, or dehydrated, using asuitable water-miscible organic solvent, and/or converted, in anappropriate manner, for intended subsequent use.
 6. A method as claimedin claim 1 wherein the procedure is carried out at a pH of from 1 to 12.7. A method as claimed in claim 1 wherein the procedure is carried outat a temperature of from 0 to 100° C.
 8. A method as claimed in claim 1wherein the polyanhydroglucuronic acid-containing material is obtainedby oxidation of a suitable polysaccharide, such as native or regeneratedcellulose or starch.
 9. Stable microdispersed polyanhydroglucuronic acidand salts thereof wherever prepared by a method as claimed in claim 1.10. Stable microdispersed polyanhydroglucuronic acid and salts thereofcontaining in their polymeric chain from 8 to 30 percent by weight ofcarboxyl groups, at least 80 percent by weight of these groups being ofthe uronic type, at most 5 percent by weight of carbonyl groups, and atmost 0.5 percent by weight of bound nitrogen.
 11. Polyanhydroglucuronicacid and salts thereof as claimed in claim 10 containing in theirpolymeric chain at most 0.2 percent by weight of bound nitrogen. 12.Polyanhydroglucuronic acid and salts thereof as claimed in claim 10wherein the molecular mass of the polymeric chain is from 1×10³ to 3×10⁵Daltons.
 13. Polyanhydroglucuronic acid and salts thereof according toclaim 12 wherein the molecular mass of the polymeric chain ranges from5×10³ to 1.5×10⁵ Daltons.
 14. Polyanhydroglucuronic acid and saltsthereof as claimed in claim 10 wherein the content of carboxyl groups isin the range of from 12 to 26 percent by weight, at least 95 percent ofthese groups being of the uronic type.
 15. Polyanhydroglucuronic acidand salts thereof as claimed in claim 10 containing at most 1 percent byweight of carbonyl groups.
 16. Polyanhydroglucuronic acid and saltsthereof according to claim 10 wherein the carbonyl groups are intra- andintermolecular 2,6 and 3,6 hemiacetals, 2,4-hemialdals and C2-C3aldehydes.
 17. Stable microdispersed polyanhydroglucuronic acid andsalts thereof according to claim 10 made up of particles sized from 0.1to 1000 μm.
 18. Stable microdispersed polyanhydroglucuronic acid andsalts thereof according to claim 10 made up of fibres of from 5 to 30 μmin diameter and up to 30 mm length.
 19. A pharmaceutical or cosmeticcomposition incorporating stable microdispersed polyanhydroglucuronicacid and salts thereof as claimed in claim
 10. 20. A composition asclaimed in claim 19 in the form of a haemostatically active aerosolcomposition.